1. Field of the Invention
The present invention is in the field of methods for the transfer of genetic information, e.g., foreign DNA, into target cells, especially eukaryotic cells. In particular, the present invention relates to nonviral gene carriers comprising multifunctional molecular conjugates which include, inter alia, lipopolyamines of a particular configuration, a component which promotes endosome disruption, and a receptor specific binding component. The present application is related to U.S. Ser. No. 08/314,060 filed Sep. 28, 1994 and entitled "Multifunctional molecular complexes for gene transfer to cells" which is incorporated herein by reference.
Heretofore, viral vectors of various types have been successfully utilized for the insertion of selected foreign genetic information into a target cell, and in the case of eukaryotic cells, for incorporation of that genetic information into the genome of the cell. These viral vector systems have relied upon the molecular machinery of the virus, evolved over time to surmount the significant problems facing a virus in attempting to invade, i.e., infect a cell. Despite the efficiency of such viral vectors, however, there has been continued concern regarding the safety of using viruses, particularly from the standpoint of undesired side effects. Thus, there has been an ongoing effort to develop non-viral gene delivery systems that are as efficient as viral vectors, but with an improved safety profile.
Nonviral vectors or carriers, of the type with which the present invention is concerned, will thus have to overcome the same obstacles as a viral vector. The problems faced by such carriers include persistence in the biophase of the organism for a sufficient time to reach the target cell; recognition of the target cell and means for mediating transport of the genetic material through the cell membrane and into the cytoplasm of the cell; avoidance of degradation within the cell by the reticuloendothelial system; and transport to and through the nuclear membrane into the nucleus of the cell where transcription of the genetic material can take place.
It is to overcoming the problems described above that the present invention is addressed; and since the problems are several and different, the present invention comprises a multifunctional complex, i.e., a molecular conjugate of various ligands intended to surmount specific obstacles.
The ultimate usefulness of gene transfer techniques is of enormous potential benefit in a number of areas. The transfer of genetic material into cells is the basis of a number of processes now widely accepted in the areas of molecular biology, gene therapy and genetic immunization. Transfer of the genetic information encoded in DNA to cells where it expresses identified individual proteins, has permitted investigation of the function of such proteins on a cellular level, and of the underlying cell physiology. Genetic material has also been transferred into cells to introduce proteins that are absent due to an inherent genetic flaw in the cell that expresses an inactive protein or else prevents expression of the protein altogether. The transfer of genetic material into cells can be used to prevent the expression of proteins in those cells through the well-known antisense effect of complementary DNA or RNA strands.
Exogenous, i.e., foreign genetic material can permit cells to synthesize significant amounts of proteins that are not available by other means in practical economic terms. These proteins of interest can be grown in a variety of host cells such as yeast, bacterial or mammalian cells. Genetic material can also be used to provide protective immune responses in vivo by injection of DNA that encodes immunogenic proteins, i.e., ones that can stimulate the desired immune response. The in vivo introduction of exogenous genetic material into cells also has potential utility in applications for the alleviation, treatment or prevention of metabolic, tumoral or infectious disorders by the same mechanisms enumerated above.
2. Description of the Prior Art
It is possible to transfer genetic material into target cells without the use of vectors or carriers. For example, genetic material can be introduced systemically through an intravenous or intraperitoneal injection for in vivo applications, or can be introduced to the site of action by direct injection into that area. For example, it has long been recognized that DNA, by itself, injected into various tissues, will enter cells and produce a protein that will elicit an immune response. See, e.g., P. Atanasiu et al., Academie des Sciences (Paris) 254, 4228-30 (1962); M. A. Israel et al., J. Virol. 29, 990-96 (1979); H. Will et al., Nature, 299, 740-42 (1982); H. Robinson, World Patent Application WO 86/00930, published Feb. 13, 1986; P. L. Felgner, J. A. Wolff, G. H. Rhodes, R. w. Malone and D. A. Carson, World Patent Application WO 90/11092, published Oct. 4, 1990; and R. J. Debs and N. Zhu, World Patent Application WO 93/24640 published Dec. 9, 1993. However, DNA by itself is hydrophilic, and the hydrophobic character of the cellular membrane poses a significant barrier to the transfer of DNA across it. Accordingly, it has become preferred in the art to use facilitators that enhance the transfer of DNA into cells on direct injection.
Another approach in the art to delivery of genetic material to target cells is one that takes advantage of natural receptor-mediated endocytosis pathways that exist in such cells. Several cellular receptors have been identified heretofore as desirable agents by means of which it is possible to achieve the specific targeting of drugs, and especially macromolecules and molecular conjugates serving as carriers of genetic material of the type with which the present invention is concerned. These cellular receptors allow for specific targeting by virtue of being localized to a particular tissue or by having an enhanced avidity for, or activity in a particular tissue. See, e.g., J. L. Bodmer and R. T. Dean, Meth. Enzymol., 112, 298-306 (1985). This affords the advantages of lower doses or significantly fewer undesirable side effects.
One of the better known examples of a cell and tissue selective receptor is the asialoglycoprotein receptor present in hepatocytes. The asialoglycoprotein receptor is an extracellular receptor with a high affinity for galactose, especially tri-antennary oligosaccharides, i.e., those with three somewhat extended chains or spacer arms having terminal galactose residues; see, e.g., H. F. Lodish, TIBS, 16, 374-77 (1991). This high affinity receptor is localized to hepatocytes and is not present in Kupffer cells; allowing for a high degree of selectivity in delivery to the liver.
It has also been proposed in the art of receptor-mediated gene transfer that in order for the process to be efficient in vivo, the assembly of the DNA complex should result in condensation of the DNA to a size suitable for uptake via an endocytic pathway. See, e.g., J. C. Perales, T. Ferkol, H. Beegen, O. D. Ratnoff, and R. W. Hanson, Proc. Nat. Acad. Sci. USA, 91, 4086-4090 (1994).
An alternative method of providing cell-selective binding is to attach an entity with an ability to bind to the cell type of interest; commonly used in this respect are antibodies which can bind to specific proteins present in the cellular membranes or outer regions of the target cells. Alternative receptors have also been recognized as useful in facilitating the transport of macromolecules, such as biotin and folate receptors; see P. S. Low, M. A. Horn and P. F. Heinstein, World Patent Application WO 90/12095, published Oct. 18, 1990; P. S. Low, M. A. Horn and P. F. Heinstein, World Patent Application WO 90/12096, published Oct. 18, 1990; P. S. Low, M. A. Horn and P. F. Heinstein, U.S. Pat. No. 5,108,921, Apr. 28, 1992; C. P. Leamon and P. S. Low, Proc. Nat. Acad. Sci. USA, 88, 5572-5576 (1991); transferrin receptors; insulin receptors; and mannose receptors (see further below). The enumerated receptors are merely representative, and other examples will readily come to the mind of the artisan.
The conjugation of different functionalities on the same molecule has also been utilized in the art. For example, in 1988 G. Y. Wu and C. M. Wu, J. Biol. Chem., 263, 14621-14624 (1988) described a method for cellular receptor mediated delivery of DNA to hepatocytes. This method was further described in G. Y. Wu and C. H. Wu, Biochem., 27, 887-892 (1988) ; G. Y. Wu and C. H. Wu, U.S. Pat. No. 5,166,320, Nov. 24, 1992; and G. Y. Wu and C. H. Wu, World Patent Application WO 92/06180, published Apr. 16, 1992. The method consists of attaching a glycoprotein, asialoorosomucoid, to poly-lysine to provide a hepatocyte selective DNA carrier. The function of the poly-lysine is to bind to the DNA through ionic interactions between the positively charged (cationic) .epsilon. amino groups of the lysines and the negatively charged (anionic) phosphate groups of the DNA. Orosomucoid is a glycoprotein which is normally present in human serum. Removal of the terminal sialic acid (N-acetyl neuraminic acid) from the branched oligosaccharides exposes terminal galactose oligosaccharides, for which hepatocyte receptors have a high affinity, as already described.
After binding to the asialoglycoprotein receptor on hepatocytes, the protein is taken into the cell by endocytosis into a pre-lysosomal endosome. The DNA, lonically bound to the poly-lysine-asialoorosomucoid carrier, is also taken into the endosome. Additional work using this delivery system, e.g., that done by J. M. Wilson, M. Grossman, J. A. Cabrera, C. H. Wu and G. Y. Wu, J. Biol. Chem, 5 267, 11483-11489 (1992), has found that partial hepatectomy improves the persistence of the expression of the DNA delivered into the hepatocytes. The transfer of the DNA into cells by this mechanism is also significantly enhanced by the addition of cationic lipids; see, e.g., K. D. Mack, R. Walzem and J. B. Zeldis, Am. J. Med. Sci., 307, 138-143 (1994).
The use of a specific asialoglycoprotein is not required to achieve binding to the asialoglycoprotein receptor; this binding can also be accomplished with high affinity by the use of small, synthetic molecules having a similar configuration. The carbohydrate portion can be removed from an appropriate glycoprotein and be conjugated to other macromolecules; see, e.g., S. J. Wood and R. Wetzel, Bioconj. Chem., 3, 391-396 (1992). By this procedure the cellular receptor binding portion of the glycoprotein is removed, and the specific portion required for selective cellular binding can be transferred to another molecule.
There is a ample literature on the preparation of synthetic glycosides which can be attached to macromolecules and confer on them the ability to bind to the corresponding galactose specific receptor. The importance of branched glycosides was recognized early; see Y. C. Lee, Carb. Res., 67, 509-514 (1978). Further work delineated that sugar density [K. Kawaguchi, M. Kuhlenschmidt, S. Roseman and Y. C. Lee, J.Biol. Chem., 256,2230-2234 (1981)] and spacial relationships [Y. C. Lee, R. R. Townsend, M. R. Hardy, J. Lonngren, J. Arnarp, M. Haraldsson and H. Lonn, J. Biol. Chem., 258, 199-202 (1983)] are important determinants of binding potency. Reductive amination of a peptide with a branched tri-lysine amino terminus gives a ligand ending with four galactosyl residues that can be readily coupled to poly-lysine or other macromolecules; see C. Plank, K. Zatlouhal, M. Cotten, K. Mechtler and E. Wagner, Bioconj. Chem., 3,533-539(1992); and has been used to prepare DNA constructs.
Thiopropionate and thiohexanoate glycosidic derivatives of galactose have been prepared and linked to L-lysyl-L-lysine to form a synthetic tri-antennary galactose derivative. A bisacridine spermidine derivative containing this synthetic tri-antennary galactose has been used to target DNA to hepatocytes; see F. C. Szoka, Jr and J. Haensler, World Pat Application WO 93/19768, published Oct. 14,1993; and J. Haensler and F. C. Szoka, Jr., Bioconj. Chem., 4, 85-93 (1993).
Other means of providing cellular receptor based facilitation of gene transfer into cells using poly-lysine as a carrier have been described in the art. Antibodies specific for cell surface thrombomodulin have been used with poly-lysine as a delivery system for DNA in vitro and in vivo; see V. S. Trubetskoy, V. P. Torchilin, S. J. Kennel and L. Huang, Bioconj. Chem., 3, 323-327 (1992). The transferrin receptor has also been used to target DNA to erythroblasts, K562 macrophages and ML-60 leukemic cells; see E. Wagner, M. Zenke, M. Cotten, H. Beug and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA, 87, 3410-3414 (1990); M. Zenke, P. Steinlein, E. Wagner, M. Cotten, H. Beug and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA, 87, 3655-3659 (1990); and G. Citro, D. Perrotti, C. Cucco, I. D'Agnano, A. Sacchi, G. Zupi and B. Calabretta, Proc. Nat. Acad. Sci. USA, 89, 7031-7035 (1990). These studies used both small oliogodeoxynucleotides as well as large plasmids.
The ability of poly-lysine to facilitate DNA entry into cells is significantly enhanced if the poly-lysine is chemically modified with hydrophobic appendages; see X. Zhou and L. Huang, Biochim. Biophys. Acta, 1189, 195-203 (1994); complexed with cationic lipids; see K. D. Mack, R. Walzem and J. B. Zeldis, Am. J. Med. Sci., 307, 138-143 (1994) or associated with viruses. Many viruses infect specific cells by receptor mediated binding and insertion of the viral DNA/RNA into the cell; and thus this action of the virus is similar to the facilitated entry of DNA described above.
Replication-incompetent adenovirus has been used to enhance the entry of transferrin-poly-lysine complexed DNA into cells; see D. T. Curiel, S. Agarwal, E. Wagner and M. Cotten, Proc. Nat. Acad. Sci. USA, 88, 8850-8854 (1991); E. Wagner, K. Zatloukal, M. Cotten, H. Kirlappos, K. Mechtler, D. T. Curiel and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA, 89, 6099-6103 (1992); M. Cotten, E. Wagner, K. Zatloukal, S. Phillips, D. T. Curiel and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA, 89, 6094-6098 (1992); and L. Gao, E. Wagner, M. Cotten, S. Agarwal, C. Harris, M. Romer, L. Miller, P.-C. Hu and D. Curiel, Hum. Gene Ther., 4, 17-24 (1993). The adenovirus enhances the entry of the poly-lysine-transferrin-DNA complex when covalently attached to the poly-lysine and when attached through an antibody binding site. There does not need to be a direct attachment of the adenovirus to the poly-lysine-transferrin-DNA complex, and it can facilitate the entry of the complex when present as a simple mixture. The poly-lysine transferrin-DNA complex provides receptor specific binding to the cells and is internalized into endosomes along with the DNA. Once inside the endosomes, the adenovirus facilitates entry of the DNA/transferrin-poly-lysine complex into the cell by disruption of the endosomal compartment with subsequent release of the DNA into the cytoplasm. Replication-incompetent adenovirus has also been used to enhance the entry of uncomplexed DNA plasmids into cells without the benefit of the cell receptor selectivity conferred by the poly-lysine-transferrin complex; see K. Yoshimura, M. A. Rosenfeld, P. Seth and R. G. Crystal, J. Biol. Chem., 268, 2300-2303 (1993).
Synthetic peptides such as the N-terminus region of the influenza hemagglutinin protein are known to destabilize membranes and are known as fusogenic peptides. Conjugates containing the influenza fusogenic peptide coupled to poly-lysine together with a peptide having a branched tri-lysine amino terminus ligand ending with four galactosyl residues have been prepared as facilitators of DNA entry into hepatocytes; see C. Plank, K. Zatlouhal, M. Cotten, K. Mechtler and E. Wagner, Bioconj. Chem., 3,533 -539 (1992). These conjugates combine the asialoglycoprotein receptor mediated binding conferred by the tetra-galactose peptide, the endosomal disrupting abilities of the influenza fusogenic peptide, and the DNA binding of the poly-lysine. These conjugates deliver DNA into the cell by a combination of receptor mediated uptake and internalization into endosomes. This internalization is followed by disruption of the endosomes by the influenza fusogenic peptide to release the DNA into the cytoplasm. In a similar fashion, the influenza fusogenic peptide can be attached to poly-lysine and mixed with the transferrin-poly-lysine complex to provide a similar DNA carrier selective for cells carrying the transferrin receptor; see E. Wagner, C. Plank, K. Zatloukal, M. Cotten and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA, 89, 7934-7938 (1992). Synthetically designed peptides can also be used; for example the "GALA" peptides [N. K. Subbarao, R. A. Parente, F. C. Szoka, Jr, L. Nadasdi and K. Pongracz, J. Biol. Chem., 26, 2964-2972 (1987)] have been coupled to DNA carriers and an enhanced facilitated entry into cells was observed [J. Haensler and F. C. Szoka, Jr., Bioconj. Chem., 4, 372-379 (1993)]. The cationic amphipathic peptide gramicidin S can facilitate entry of DNA into cells [J. -Y. Legendre and F. C. Szoka, Jr., Proc. Nat. Acad. Sci. USA, 90, 893-897 (1993)], but also requires a phospholipid to achieve significant transfer of DNA.
Poly-lysine is not unique in providing a polycationic framework for the entry of DNA into cells. DEAE-dextran has also been shown to be effective in promoting RNA and DNA entry into cells; see R. Juliano and E. Mayhew, Exp. Cell. Res. 73, 3-12 (1972); and E. Mayhew and R. Juliano, Exp. Cell. Res. 77, 409-414 (1973). More recently, a dendritic cascade co-polymer of ethylenediamine and methyl acrylate has been shown to be useful in providing a carrier of DNA which facilitates entry into cells; see J. Haensler and F. C. Szoka,Jr., Bioconj. Chem., 4, 372-379 (1993). An alkylated polyvinylpyridine polymer has also been used to facilitate DNA entry into cells; see A. V. Kabanov, I. V. Astafieva, I. V. Maksimova, E. M. Lukanidin, G. P. Georgiev and V. A. Kabanov, Bioconj. Chem., 4, 448-454 (1993).
Positively charged liposomes have also been widely used as carriers of DNA which facilitate entry into cells; see, e.g., F. C. Szoka,Jr. and J. Haensler, World Pat Application WO 93/19768, published Oct. 14, 1993; R. J. Debs and N. Zhu, World Patent Application WO 93/24640, published Dec. 9, 1993; P. L. Felgner, R. Kumar, C. Basava, R. C. Border and J.-Y. Hwang-Felgner, World Patent Application WO 15 91/16024, published Oct. 31, 1991; P. L. Felgner and G. M. Ringold, Nature, 337, 387-388 (1989); J. K. Rose, L. Buonocore and M. A. Whitt, BioTechniques, 10, 520-525 (1991); C. F. Bennett, M. Y. Chiang, H. Chan, J. E. E. Schoemaker and C. K. Mirabelli, Mol. Pharm. 41, 1023-1033 (1992); J. H. Felgner, R. Kumar, C. N. Sridhar, C. J. Wheeler, Y. J. Tsai, R. Border, P. Ramsey, M. Martin and P. L. Felgner, J. Biol. Chem., 269, 2550-2561 (1994); J. G. Smith, R. L. Walzem and J. B. German, Biochim. Biophys. Acta, 1154, 327-340 (1993). These carrier compositions have also included pH sensitive liposomes; see C.-J. Chu, J. Dijkstra, M.-Z. Lai, K. Hong and F. C. Szoka,Jr., Pharm. Res., 7, 824-854 (1990); J.-Y. Legendre and F. C. Szoka,Jr., Pharm. Res., 9, 1253-1242 (1992).
A poly-cationic lipid has been prepared by coupling dioctadecylamidoglycine and dipalmitoyl phosphatidylethanolamine to a 5-carboxyspermine; see J.-P. Behr, B. Demeniex, J.-P.Loeffler and J. Perez-Mutul, Proc. Nat. Acad. Sci. USA, 86, 6982-6986 (1989); F. Barthel, J.-S. Remy, J.-P.Loeffler and J. P. Behr, DNA and Cell Biol., 12, 553-560 (1993); J.-P. Loeffler and J.-P. Behr, Meth. Enzymol., 35 217, 599-618 (1993); J.-P. Behr and J.-P. Loeffler, U.S. Pat. No. 5,171,678, Dec. 15, 1992. These lipophilic-spermines are very active in transferring DNA through cellular membranes.
Combinations of lipids have been used to facilitate the transfer of nucleic acids into cells. For example, in U.S. Pat. No. 5,283,185 there is disclosed such a method which utilizes a mixed lipid dispersion of a cationic lipid with a co-lipid in a suitable solvent. The lipid has a structure which includes a lipophilic group derived from chlolesterol, a linker bond, a linear alkyl spacer arm, and a cationic amino group; and the co-lipid is phosphatidylcholine or phosphatidylethanolamine.
Macrophages have receptors for both mannose and mannose-6-phosphate which can bind to and internalize molecules displaying these sugars. The molecules are internalized by endocytosis into a pre-lysosomal endosome. This internalization has been used to enhance entry of oligonucleotides into macrophages using bovine serum albumin modified with mannose-6-phosphate and linked to an oligodeoxynucleotide by a disulfide bridge to a modified 3' end; see E. Bonfils, C. Depierreux, P. Midoux, N. T. Thuong, M. Monsigny and A. C. Roche, Nucl. Acids Res. 20, 4621-4629 (1992). Similarly, oligodeoxynucleotides modified at the 3' end with biotin were combined with mannose-modified streptavidin, and were also found to have facilitated entry into macrophages; see E. Bonfils, C. Mendes, A. C. Roche, M. Monsigny and P. Midoux, Bioconj. Chem., 3, 277-284 (1992).
Various peptides and proteins, many of which are naturally occurring, have been shown to have receptors on cell surfaces, that once they are attached thereto, allow them to become internalized by endocytosis. Materials bound to these receptors are delivered to endosomal compartments inside the cell. Examples include insulin, vasopressin, low density lipoprotein, epidermal growth factor and others. This internalization has also been used to facilitate entry of DNA into cells; e.g., insulin has been conjugated to polylysine to provide facilitated DNA entry into cells possessing an insulin receptor; see B. Huckett, M. Ariatti and A. O. Hawtrey, Biochem. Pharmacol., 40, 253-263 (1990).